Methods for making a device for concurrently processing multiple biological chip assays

ABSTRACT

Methods for concurrently processing multiple biological chip assays by providing a biological chip plate comprising a plurality of test wells, each test well having a biological chip having a molecular probe array; introducing samples into the test wells; subjecting the biological chip plate to manipulation by a fluid handling device that automatically performs steps to carry out reactions between target molecules in the samples and probes; and subjecting the biological chip plate to a biological chip plate reader that interrogates the probe arrays to detect any reactions between target molecules and probes.

BACKGROUND OF THE INVENTION

This invention relates to methods for concurrently performing multiplebiological chip assays. The invention therefore relates to diversefields impacted by the nature of molecular interaction, includingchemistry, biology, medicine and diagnostics.

New technology, called VLSIPS™, has enabled the production of chipssmaller than a thumbnail that contain hundreds of thousands or more ofdifferent molecular probes. These biological chips or arrays have probesarranged in arrays, each probe assigned a specific location. Biologicalchips have been produced in which each location has a scale of, forexample, ten microns. The chips can be used to determine whether targetmolecules interact with any of the probes on the chip. After exposingthe array to target molecules under selected test conditions, scanningdevices can examine each location in the array and determine whether atarget molecule has interacted with the probe at that location.

Biological chips or arrays are useful in a variety of screeningtechniques for obtaining information about either the probes or thetarget molecules. For example, a library of peptides can be used asprobes to screen for drugs. The peptides can be exposed to a receptor,and those probes that bind to the receptor can be identified.

Arrays of nucleic acid probes can be used to extract sequenceinformation from, for example, nucleic acid samples. The samples areexposed to the probes under conditions that allow hybridization. Thearrays are then scanned to determine to which probes the samplemolecules have hybridized. One can obtain sequence information bycareful probe selection and using algorithms to compare patterns ofhybridization and non-hybridization. This method is useful forsequencing nucleic acids, as well as sequence checking. For example, themethod is useful in diagnostic screening for genetic diseases or for thepresence and/or identity of a particular pathogen or a strain ofpathogen. For example, there are various strains of HIV, the virus thatcauses AIDS. Some of them have become resistant to current AIDStherapies. Diagnosticians can use DNA arrays to examine a nucleic acidsample from the virus to determine what strain it belongs to.

Currently, chips are treated individually, from the step of exposure tothe target molecules to scanning. These methods yield exquisitelydetailed information. However, they are not adapted for handlingmultiple samples simultaneously. The ability to do so would beadvantageous in settings in which large amounts of information arerequired quickly, such as in clinical diagnostic laboratories or inlarge-scale undertakings such as the Human Genome Project.

SUMMARY OF THE INVENTION

This invention provides methods for concurrently processing multiplebiological chip assays. According to the methods, a biological chipplate comprising a plurality of test wells is provided. Each test welldefines a space for the introduction of a sample and contains abiological array. The array is formed on a surface of the substrate,with the probes exposed to the space. A fluid handling devicemanipulates the plates to perform steps to carry out reactions betweenthe target molecules in samples and the probes in a plurality of testwells. The biological chip plate is then interrogated by a biologicalchip plate reader to detect any reactions between target molecules andprobes in a plurality of the test wells, thereby generating results ofthe assay. In a further embodiment of the invention, the method alsoincludes processing the results of the assay with a computer. Suchanalysis is useful when sequencing a gene by a method that uses analgorithm to process the results of many hybridization assays to providethe nucleotide sequence of the gene.

The methods of the invention can involve the binding of tagged targetmolecules to the probes. The tags can be, for example, fluorescentmarkers, chemiluminescent markers, light scattering markers orradioactive markers. In certain embodiments, the probes are nucleicacids, such as DNA or RNA molecules. The methods can be used to detector identify a pathogenic organism, such as HIV, or to detect a humangene variant, such a the gene for a genetic disease such as cysticfibrosis, diabetes, muscular dystrophy-or predisposition to certaincancers.

This invention also provides systems for performing the methods of thisinvention. The systems include a biological chip plate; a fluid handlingdevice that automatically performs steps to carry out assays on samplesintroduced into a plurality of the test wells; a biological chip platereader that determines in a plurality of the test wells the results ofthe assay and,.optionally, a computer comprising a program forprocessing the results. The fluid handling device and plate reader canhave a heater/cooler controlled by a thermostat for controlling thetemperature of the samples in the test wells and robotically controlledpipets for adding or removing fluids from the test wells atpredetermined times.

In certain embodiments, the probes are attached by light-directed probesynthesis. The biological chip plates can have 96 wells arranged in 8rows and 12 columns, such as a standard microtiter plate. The probearrays can each have at least about 100, 1000, 100,000 or 1,000,000addressable features (e.g., probes). A variety of probes can be used onthe plates, including, for example, various polymers such as peptides ornucleic acids.

The plates can have wells in which the probe array in each test well isthe same. Alternatively, when each of several samples are to besubjected to several tests, each row can have the same probe array andeach column can have a different array. Alternatively, all the wells canhave different arrays.

Several methods of making biological chip plates are contemplated. Inone method, a wafer and a body are provided. The wafer includes asubstrate and a surface to which is attached a plurality of arrays ofprobes. The body has a plurality of channels. The body is attached tothe surface of the wafer whereby the channels each cover an array ofprobes and the wafer closes one end of a plurality of the channels,thereby forming test wells defining spaces for receiving samples. In asecond method, a body having a plurality of wells defining spaces isprovided and biological chips are provided. The chips are attached tothe wells so that the probe arrays are exposed to the space. Anotherembodiment involves providing a wafer having a plurality of probearrays; and applying a material resistant to the flow of a liquid sampleso as to surround the probe arrays, thereby creating test wells.

This invention also provides a wafer for making a biological chip plate.The wafer has a substrate and a surface to which are attached aplurality of probe arrays. The probe arrays are arranged on the wafersurface in rows and columns, wherein the probe arrays in each row arethe same and the probe arrays in each column are different.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a system of this invention having a biological chipplate, fluid handling device, biological chip plate reader and computer;

FIG. 2 depicts the scanning of a biological chip plate by a biologicalchip plate reader;

FIG. 3 depicts a biological plate of this invention;

FIG. 4 depicts the mating of a wafer containing many biological arrayswith a body having channels to create a biological chip plate;

FIG. 5 depicts a biological chip plate in cross section having a bodyattached to a wafer to create closed test wells in which a probe arrayis exposed to the space in the test well;

FIG. 6 depicts a biological plate in cross section having a body whichhas individual biological chips attached to the bottom of the wells;

FIG. 7 is a top-down view of a test well containing a biological array;and

FIG. 8 depicts a method of producing an array of oligonucleotide probeson the surface of a substrate by using a mask to expose certain parts ofthe surface to light, thereby removing photoremovable protective groups,and attaching nucleotides to the exposed reactive groups.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The following terms are intended to have the following general meaningsas they are used herein:

A. Complementary:

Refers to the topological compatibility or matching together ofinteracting surfaces of a probe molecule and its target. Thus, thetarget and its probe can be described as complementary, and furthermore,the contact surface characteristics are complementary to each other.

B. Probe:

A probe is a surface-immobilized molecule that can be recognized by aparticular target. Examples of probes that can be investigated by thisinvention include, but are not restricted to, agonists and antagonistsfor cell membrane receptors, toxins and venoms, viral epitopes, hormones(e.g., opioid peptides, steroids, etc.), hormone receptors, peptides,enzymes, enzyme substrates, cofactors, drugs, lectins, sugars,oligonucleotides, nucleic acids, oligosaccharides proteins, andmonoclonal antibodies.

C. Target:

A molecule that has an affinity for a given probe. Targets may benaturally-occurring or man-made molecules. Also, they can be employed intheir unaltered state or as aggregates with other species. Targets maybe attached, covalently or noncovalently, to a binding member, eitherdirectly or via a specific binding substance. Examples of targets whichcan be employed by this invention include, but are not restricted to,antibodies, cell membrane receptors, monoclonal antibodies and antiserareactive with specific antigenic determinants (such as on viruses, cellsor other materials), drugs, oligonucleotides, nucleic acids, peptides,cofactors, lectins, sugars, polysaccharides, cells, cellular membranes,and organelles. Targets are sometimes referred to in the art asanti-probes. As the term targets is used herein, no difference inmeaning is intended. A “Probe Target Pair” is formed when twomacromolecules have combined through molecular recognition to form acomplex.

D. Array:

A collection of probes, at least two of which are different, arranged ina spacially defined and physically addressable manner.

E. Biological Chip:

A substrate having a surface to which one or more arrays of probes isattached. The substrate can be, merely by way of example, silicon orglass and can have the thickness of a glass microscope slide or a glasscover slip. Substrates that are transparent to light are useful when themethod of performing an assay on the chip involves optical detection. Asused herein, the term also refers to a probe array and the substrate towhich it is attached that form part of a wafer.

F. Wafer:

A substrate having a surface to which a plurality of probe arrays areattached. On a wafer, the arrays are physically separated by a distanceof at least about a millimeter, so that individual chips can be made bydicing a wafer or otherwise physically separating the array into unitshaving a probe array.

G. Biological Chip Plate:

A device having an array of biological chips in which the probe array ofeach chip is separated from the probe array of other chips by a physicalbarrier resistant to the passage of liquids and forming an area orspace, referred to as a “test well,” capable of containing liquids incontact with the probe array.

II. General

This invention provides automated methods for concurrently processingmultiple biological chip assays. Currently available methods utilizeeach biological chip assay individually. The methods of this inventionallow many tests to be set up and processed together. Because they allowmuch higher throughput of test samples, these methods greatly improvethe efficiency of performing assays on biological chips.

In the methods of this invention, a biological chip plate is providedhaving a plurality of test wells. Each test well includes a biologicalchip. Test samples, which may contain target molecules, are introducedinto the test wells. A fluid handling device exposes the test wells to achosen set of reaction conditions by, for example, adding or removingfluid from the wells, maintaining the liquid in the wells atpredetermined temperatures, and agitating the wells as required, therebyperforming the test. Then, a biological chip reader interrogates theprobe arrays in the test wells, thereby obtaining the results of thetests. A computer having an appropriate program can further analyze theresults from the tests.

Referring to FIG. 1, one embodiment of the invention is a system forconcurrently processing biological chip assays. The system includes abiological chip plate reader 100, a fluid handling device 110, abiological chip plate 120 and, optionally, a computer 130. In operation,samples are placed in wells on the chip plate 120 with fluid handlingdevice 110. The plate optionally can be moved with a stage translationdevice 140. Reader 100 is used to identify where targets in the wellshave bound to complementary probes. The system operates under control ofcomputer 130 which may optionally interpret the results of the assay.

A. Biological Chip Plate Reader

In assays performed on biological chips, detectably labeled targetmolecules bind to probe molecules. Reading the results of an assayinvolves detecting a signal produced by the detectable label. Readingassays on a biological chip plate requires a biological chip reader.Accordingly, locations at which target(s) bind with complementary probescan be identified by detecting the location of the label. Throughknowledge of the characteristics/sequence of the probe versus location,characteristics of the target can be determined. The nature of thebiological chip reader depends upon the particular type of labelattached to the target molecules.

The interaction between targets and probes can be characterized in termsof kinetics and thermodynamics. As such, it may be necessary tointerrogate the array while in contact with a solution of labeledtargets. In such systems, the detection system must be extremelyselective, with the capacity to discriminate between surface-bound andsolution-born targets. Also, in order to perform a quantitativeanalysis, the high-density of the probe sequences requires the system tohave the capacity to distinguish between each feature site. The systemalso should have sensitivity to low signal and a large dynamic range.

In one embodiment, the chip plate reader includes a confocal detectiondevice having a monochromatic or polychromatic light source, a focusingsystem for directing an excitation light from the light source to thesubstrate, a temperature controller for controlling the substratetemperature during a reaction, and a detector for detecting fluorescenceemitted by the targets in response to the excitation light. The detectorfor detecting the fluorescent emissions from the substrate, in someembodiments, includes a photomultiplier tube. The location to whichlight is directed may be controlled by, for example, an x-y-ztranslation table. Translation of the x-y-z table, temperature control,and data collection are managed and recorded by an appropriatelyprogrammed digital computer.

Further details for methods of detecting fluorescently labelledmaterials on biological chips are provided in U.S. patent applicationSer. No. 08/195,889, filed Feb. 10, 1994 and incorporated herein byreference.

FIG. 2 illustrates the reader according to one specific embodiment. Thechip plate reader comprises a body 200 for immobilizing the biologicalchip plate: Excitation radiation, from an excitation source 210 having afirst wavelength, passes through excitation optics 220 from below thearray. The light passes through the chip plate since it is transparentto at least this wavelength of light. The excitation radiation excites aregion of a probe array on the biological chip plate 230. In response,labeled material on the sample emits radiation which has a wavelengththat is different from the excitation wavelength. Collection optics 240,also below the array, then collect the emission from the sample andimage it onto a detector 250, which can house a CCD array, as describedbelow. The detector generates a signal proportional to the amount ofradiation sensed thereon. The signals can be assembled to represent animage associated with the plurality of regions from which the emissionoriginated.

According to one embodiment, a multi-axis translation stage 260 movesthe biological chip plate to position different wells to be scanned, andto allow different probe portions of a probe array to be interrogated.As a result, a 2-dimensional image of the probe arrays in each well isobtained.

The biological chip reader can include auto-focusing feature to maintainthe sample in the focal plane of the excitation light throughout thescanning process. Further, a temperature controller may be employed tomaintain the sample at a specific temperature while it is being scanned.The multi-axis translation stage, temperature controller, auto-focusingfeature, and electronics associated with imaging and data collection aremanaged by an appropriately programmed digital computer 270.

In one embodiment, a beam is focused onto a spot of about 2 μm indiameter on the surface of the plate using, for example, the objectivelens of a microscope or other optical means to control beam diameter.(See, e.g., U.S. patent application Ser. No. 08/195,889, supra.)

In another embodiment, fluorescent probes are employed in combinationwith CCD imaging systems. Details of this method are described in U.S.application Ser. No. 08/301,051, incorporated herein by reference in itsentirely. In many commercially available microplate readers, typicallythe light source is placed above a well, and a photodiode detector isbelow the well. In the present invention, the light source can bereplaced with a higher power lamp or laser. In one embodiment, thestandard absorption geometry is used, but the photodiode detector isreplaced with a CCD camera and imaging optics to allow rapid imaging ofthe well. A series of Raman holographic or notch filters can be used inthe optical path to eliminate the excitation light while allowing theemission to pass to the detector. In a variation of this method, a fiberoptic imaging bundle is utilized to bring the light to the CCD detector.In another embodiment, the laser is placed below the biological chipplate and light directed through the transparent wafer or base thatforms the bottom of the biological chip plate. In another embodiment,the CCD array is built into the wafer of the biological chip plate.

The choice of the CCD array will depend on the number of probes in eachbiological array. If 2500 probes nominally arranged in a square (50×50)are examined, and 6 lines in each feature are sampled to obtain a goodimage, then a CCD array of 300×300 pixels is desirable in this area.However, if an individual well has 48,400 probes (220×220) then a CCDarray with 1320×1320 pixels is desirable. CCD detectors are commerciallyavailable from, e.g., Princeton Instruments, which can meet either ofthese requirements.

In another embodiment, the detection device comprises a line scanner, asdescribed in U.S. patent application Ser. No. 08/301,051, filed Sep. 2,1994, incorporated herein by reference. Excitation optics focusesexcitation light to a line at a sample, simultaneously scanning orimaging a strip of the sample. Surface bound labeled targets from thesample fluoresce in response to the light. Collection optics image theemission onto a linear array of light detectors. By employing confocaltechniques, substantially only emission from the light's focal plane isimaged. Once a strip has been scanned, the data representing the1-dimensional image are stored in the memory of a computer. According toone embodiment, a multi-axis translation stage moves the device at aconstant velocity to continuously integrate and process data.Alternatively, galvometric scanners or rotating polyhedral mirrors maybe employed to scan the excitation light across the sample. As a result,a 2-dimensional image of the sample is obtained.

In another embodiment, collection optics direct the emission to aspectrograph which images an emission spectrum onto a 2-dimensionalarray of light detectors. By using a spectrograph, a full spectrallyresolved image of the sample is obtained.

The read time for a full microtiter plate will depend on thephotophysics of the fluorophore (i.e. fluorescence quantum yield andphotodestruction yield) as well as the sensitivity of the detector. Forfluorescein, sufficient signal-to-noise to read a chip image with a CCDdetector can be obtained in about 30 seconds using 3 mW/cm² and 488 nmexcitation from an Ar ion laser or lamp. By increasing the laser power,and switching to dyes such as CY3 or CY5 which have lowerphotodestruction yields and whose emission more closely matches thesensitivity maximum of the CCD detector, one easily is able to read eachwell in less than 5 seconds. Thus, an entire plate could be examinedquantitatively in less than 10 minutes, even if the whole plate has over4.5 million probes.

A computer can transform the data into another format for presentation.Data analysis can include the steps of determining, e.g., fluorescentintensity as a function of substrate position from the data collected,removing “outliers” (data deviating from a predetermined statisticaldistribution), and calculating the relative binding affinity of thetargets from the remaining data. The resulting data can be displayed asan image with color in each region varying according to the lightemission or binding affinity between targets and probes therein.

One application of this system when coupled with the CCD imaging systemthat speeds performance of the tests is to obtain results of the assayby examining the on- or off-rates of the hybridization. In oneembodiment of this method, the amount of binding at each address isdetermined at several time points after the probes are contacted withthe sample. The amount of total hybridization can be determined as afunction of the kinetics of binding based on the amount of binding ateach time point. Thus, it is not necessary to wait for equilibrium to bereached. The dependence of the hybridization rate for differentoligonucleotides on temperature, sample agitation, washing conditions(e.g. pH, solvent characteristics, temperature) can easily be determinedin order to maximize the conditions for rate and signal-to-noise.Alternative methods are described in Fodor et al., U.S. Pat. No.5,324,633, incorporated herein by reference.

B. Fluid Handling Instruments and Assay Automation

Assays on biological arrays generally include contacting a probe arraywith a sample under the selected reaction conditions, optionally washingthe well to remove unreacted molecules, and analyzing the biologicalarray for evidence of reaction between target molecules the probes.These steps involve handling fluids. The methods of this inventionautomate these steps so as to allow multiple assays to be performedconcurrently. Accordingly, this invention employs automated fluidhandling systems for concurrently performing the assay steps in each ofthe test wells. Fluid handling allows uniform treatment of samples inthe wells. Microtiter robotic and fluid-handling devices are availablecommercially, for example, from Tecan AG.

The plate is introduced into a holder in the fluid-handling device. Thisrobotic device is programmed to set appropriate reaction conditions,such as temperature, add samples to the test wells, incubate the testsamples for an appropriate time, remove unreacted samples, wash thewells, add substrates as appropriate and perform detection assays. Theparticulars of the reaction conditions depends upon the purpose of theassay. For example, in a sequencing assay involving DNA hybridization,standard hybridization conditions are chosen. However, the assay mayinvolve testing whether a sample contains target molecules that react toa probe under a specified set of reaction conditions. In this case, thereaction conditions are chosen accordingly.

C. Biological Chip Plates

FIG. 3 depicts an example of a biological chip plate 300 used in themethods of this invention based on the standard 96-well microtiter platein which the chips are located at the bottom of the wells. Biologicalchip plates include a plurality of test wells 310, each test welldefining an area or space for the introduction of a sample, and eachtest well comprising a biological chip 320, i.e., a substrate and asurface to which an array of probes is attached, the probes beingexposed to the space. FIG. 7 shows a top-down view of a well of abiological chip plate of this invention containing a biological chip onthe bottom surface of the well.

This invention contemplates a number of embodiments of the biologicalchip plate. In a preferred embodiment, depicted in FIG. 4, thebiological chip plate includes two parts. One part is a wafer 410 thatincludes a plurality of biological arrays 420. The other part is thebody of the plate 430 that contains channels 440 that form the walls ofthe well, but that are open at the bottom. The body is attached to thesurface of the wafer so as to close one end of the channels, therebycreating wells. The walls of the channels are placed on the wafer sothat each surrounds and encloses the probe array of a biological array.FIG. 5 depicts a cross-section of this embodiment, showing the wafer 510having a substrate 520 (preferably transparent to light) and a surface530 to which is attached an array of probes 540. A channel wall 550covers a probe array on the wafer, thereby creating well spaces 560.The-wafer can be attached to the body by any attachment means known inthe art, for example, gluing (e.g., by ultraviolet-curing epoxy orvarious sticking tapes), acoustic welding, sealing such as vacuum orsuction sealing, or even by relying on the weight of the body on thewafer to resist the flow of fluids between test wells.

In another preferred embodiment, depicted in cross section in FIG. 6,the plates include a body 610 having pre-formed wells 620, usuallyflat-bottomed. Individual biological chips 630 are attached to thebottom of the wells so that the surface containing the array of probes640 is exposed to the well space where the sample is to be placed.

In another embodiment, the biological chip plate has a wafer having aplurality of probe arrays and a material resistant to the flow of aliquid sample that surrounds each probe array. For example, in anembodiment useful for testing aqueous-based samples, the wafer can bescored with waxes, tapes or other hydrophobic materials in the spacesbetween the arrays, forming cells that act as test wells. The cells thuscontain liquid applied to an array by resisting spillage over thebarrier and into another cell. If the sample contains a non-aqueoussolvent, such as an alcohol, the material is selected to be resistant tocorrosion by the solvent.

The microplates of this invention have a plurality of test wells thatcan be arrayed in a variety of ways. In one embodiment, the plates havethe general size and shape of standard-sized microtiter plates having 96wells arranged in an 8×12 format. One advantage of this format is thatinstrumentation already exists for handling and reading assays onmicrotiter plates. Therefore, using such plates in biological chipassays does not involve extensive re-engineering of commerciallyavailable fluid handling devices. However, the plates can have otherformats as well.

The material from which the body of the biological chip plate is madedepends upon the use to which it is to be put. In particular, thisinvention contemplates a variety of polymers already used for microtiterplates including, for example, (poly)tetrafluoroethylene,(poly)vinylidenedifluoride, polypropylene, polystyrene, polycarbonate,or combinations thereof. When the assay is to be performed by sending anexcitation beam through the bottom of the plate collecting data throughthe bottom of the plate, the body of the plate and the substrate of thechip should be transparent to the wavelengths of light being used.

The arrangement of probe arrays in the wells of a microplate depends onthe particular application contemplated. For example, for diagnosticuses involving performing the same test on many samples, every well canhave the same array of probes. If several different tests are to beperformed on each sample, each row of the plate can have the same arrayof probes and each column can contain a different array. Samples from asingle patient are introduced into the wells of a particular column.Samples from a different patient are introduced into the wells of adifferent column. In still another embodiment, multiple patient samplesare introduced into a single well. If a well indicates a “positive”result for a particular characteristic, the samples from each patientare then rerun, each in a different well, to determine which patientsample gave a positive result.

D. Biological Chips

The biological chip plates used in the methods of this invention includebiological chips. The array of probe sequences can be fabricated on thebiological chip according to the pioneering techniques disclosed in U.S.Pat. No. 5,143,854, PCT WO 92/10092, PCT WO 90/15070, or U.S.application Ser. Nos. 08/249,188, 07/624,120, and 08/082,937,incorporated herein by reference for all purposes. The combination ofphotolithographic and fabrication techniques may, for example, enableeach probe sequence (“feature”) to occupy a very small area (“site” or“location”) on the support. In some embodiments, this feature site maybe as small as a few microns or even a single molecule. For example, aprobe array of 0.25 mm² (about the size that would fit in a well of atypical 96-well microtiter plate) could have at least 10, 100, 1000,10⁴, 10⁵ or 10⁶ features. In an alternative embodiment, such synthesisis performed according to the mechanical techniques disclosed in U.S.Pat. No. 5,384,261, incorporated herein by reference.

Referring to FIG. 8, in general, linker molecules, ^(—)O—X, are providedon a substrate. The substrate is preferably flat but may take on avariety of alternative surface configurations. For example, thesubstrate may contain raised or depressed regions on which the probesare located. The substrate and its surface preferably form a rigidsupport on which the sample can be formed. The substrate and its surfaceare also chosen to provide appropriate light-absorbing characteristics.For instance, the substrate may be functionalized glass, Si, Ge, GaAs,GAP, SiO₂, SiN₄, modified silicon, or any one of a wide variety of gelsor polymers such as (poly)tetrafluoroethylene,(poly)vinylidenedifluoride, polystyrene, polycarbonate, polypropylene,or combinations thereof. Other substrate materials will be readilyapparent to those of skill in the art upon review of this disclosure. Ina preferred embodiment the substrate is flat glass or silica.

Surfaces on the solid substrate usually, though not always, are composedof the same material as the substrate. Thus, the surface may be composedof any of a wide variety of materials, for example, polymers, plastics,resins, polysaccharides, silica or silica-based materials, carbon,metals, inorganic glasses, membranes, or any of the above-listedsubstrate materials. In one embodiment, the surface will be opticallytransparent and will have surface Si—OH functionalities, such as thosefound on silica surfaces.

A terminal end of the linker molecules is provided with a reactivefunctional group protected with a photoremovable protective group, 0-X.Using lithographic methods, the photoremovable protective group isexposed to light, hv, through a mask, M₁, that exposes a selectedportion of the surface, and removed from the linker molecules in firstselected regions. The substrate is then washed or otherwise contactedwith a first monomer that reacts with exposed functional groups on thelinker molecules (^(—)T—X). In the case of nucleic acids, the monomercan be a phosphoramidite activated nucleoside protected at the5′-hydroxyl with a photolabile protecting group.

A second set of selected regions, thereafter, exposed to light through amask, M₂, and photoremovable protective group on the linkermolecule/protected amino acid or nucleotide is removed at the second setof regions. The substrate is then contacted with a second monomercontaining a photorenovable protective group for reaction with exposedfunctional groups. This process is repeated to selectively applymonomers until polymers of a desired length and desired chemicalsequence are obtained. Photolabile groups are then optionally removedand the sequence is, thereafter, optionally capped. Side chainprotective groups, if present, are also removed.

The general process of synthesizing probes by removing protective groupsby exposure to light, coupling monomer units to the exposed activesites, and tapping unreacted sites is referred to herein as“light-directed probe synthesis.” If the probe is an oligonucleotide,the process is referred to as “light-directed oligonucleotide synthesis”and so forth.

The probes can be made of any molecules whose synthesis involvessequential addition of units. This includes polymers composed of aseries of attached units and molecules bearing a common skeleton towhich various functional groups are added. Polymers useful as probes inthis invention include, for example, both linear and cyclic polymers ofnucleic acids, polysaccharides, phospholipids, and peptides havingeither α-, β-, or ω-amino acids, heteropolymers in which a known drug iscovalently bound to any of the above, polyurethanes, polyesters,polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylenesulfides, polysiloxanes, polyimides, polyacetates, or other polymerswhich will be apparent upon review of this disclosure. Molecules bearinga common skeleton include benzodiazepines and other small molecules,such as described in U.S. Pat. No. 5,288,514, incorporated herein byreference.

Preferably, probes are arrayed on a chip in addressable rows and columnsin which the dimensions of the chip conform to the dimension of theplate test well. Technologies already have been developed to readinformation from such arrays. The amount of information that can bestored on each plate of chips depends on the lithographic density whichis used to synthesize the wafer. For example, if each feature size isabout 100 microns oh a side, each array can have about 10,000 probeaddresses in a 1 cm² area. A plate having 96 wells would contain about192,000 probes. However, if the arrays have a feature size of 20 micronson a side, each array can have close to 50,000 probes and the platewould have over 4,800,000 probes.

The selection of probes and their organization in an array depends uponthe use to which the biological chip will be put. In one embodiment, thechips are used to sequence or re-sequence nucleic acid molecules, orcompare their sequence to a referent molecule. Re-sequencing nucleicacid molecules involves determining whether a particular molecule hasany deviations from the sequence of reference molecule. For example, inone embodiment, the plates are used to identify in a particular type ofHIV in a set of patient samples. Tiling strategies for sequence checkingof nucleic acids are described in U.S. patent application Ser. No.08/284,064 (PCT/US94/12305), incorporated herein by reference.

In typical diagnostic applications, a solution containing one or moretargets to be identified (i.e., samples from patients) contacts theprobe array. The targets will bind or hybridize with complementary probesequences. Accordingly, the probes will be selected to have sequencesdirected to (i.e., having at least some complementarity with) the targetsequences to be detected, e.g., human or pathogen sequences. Generally,the targets are tagged with a detectable label. The detectable label canbe, for example, a luminescent label, a light scattering label or aradioactive label. Accordingly, locations at which targets hybridizewith complimentary probes can be identified by locating the markers.Based on the locations where hybridization occurs, information regardingthe target sequences can be extracted The existence of a mutation may bedetermined by comparing the target sequence with the wild type.

In a preferred embodiment, the detectable label is a luminescent label.Useful luminescent labels include fluorescent labels, chemi-luminescentlabels, bio-luminescent labels, and calorimetric labels, among others.Most preferably, the label is a fluorescent label such as fluorescein,rhodamine, cyanine and so forth. Fluorescent labels include, inter alia,the commercially available fluorescein phosphoramidites such asFluoreprime (Pharmacia), Fluoredite (Millipore) and FAM (ABI). Forexample, the entire surface of the substrate is exposed to the activatedfluorescent phosphoramidite, which reacts with all of the deprotected5′-hydroxyl groups. Then the entire substrate is exposed to an alkalinesolution (eg., 50% ethylenediamine in ethanol for 1-2 hours at roomtemperature). This is necessary to remove the protecting groups from thefluorescein tag.

To avoid self-quenching interactions between fluorophores on the surfaceof a biological chip, the fluorescent tag monomer should be diluted witha non-fluorescent analog of equivalent reactivity. For example, in thecase of the fluorescein phosphoramidites noted above, a 1:20 dilution ofthe reagent with a non-fluorescent phosphoramidite such as the standard5′-DMT-nucleoside phosphoramidites, has been found to be suitable.Correction for background non-specific binding of the fluorescentreagent and other such effects can be determined by routine testing.

Useful light scattering labels include large colloids, and especiallythe metal colloids such as those from gold, selenium and titanium oxide.

Radioactive labels include, for example, ³²P. This label can be detectedby a phosphoimager. Detection of course, depends on the resolution ofthe imager. Phosophoimagers are available having resolution of 50microns. Accordingly, this label is currently useful with chips havingfeatures of that size.

E. Uses

The methods of this invention will find particular use wherever highthrough-put of samples is required. In particular, this invention isuseful in clinical settings and for sequencing large quantities of DNA,for example in connection with the Human Genome project.

The clinical setting requires performing the same test on many patientsamples. The automated methods of this invention lend themselves tothese uses when the test is one appropriately performed on a biologicalchip. For example, a DNA array can determine the particular strain of apathogenic organism based on characteristic DNA sequences of the strain.The advanced techniques based on these assays now can be introduced intothe clinic. Fluid samples from several patients are introduced into thetest wells of a biological chip plate and the assays are performedconcurrently.

In some embodiments, it may be desirable to perform multiple tests onmultiple patient samples concurrently. According to such embodiments,rows (or columns). of the microtiter plate will contain probe arrays fordiagnosis of a particular disease or trait. For example, one row mightcontain probe arrays designed for a particular cancer, while other rowscontain probe arrays for another cancer. Patient samples are thenintroduced into respective columns (or rows) of the microtiter plate.For example, one column may be used to introduce samples from patient“one,” another column for patient “two” etc. Accordingly, multiplediagnostic tests may be performed on multiple patients in parallel. Instill further embodiments, multiple patient samples are introduced intoa single well. In a particular well indicator the presence of a geneticdisease or other characteristic, each patient sample is thenindividually processed to identify which patient exhibits that diseaseor trait. For relatively rarely occurring characteristics, furtherorder-of-magnitude efficiency may be obtained according to thisembodiment.

Particular assays that will find use in automation include thosedesigned specifically to detect or identify particular variants of apathogenic organism, such as HIV. Assays to detect or identify a humanor animal gene are also contemplated. In one embodiment, the assay isthe detection of a human gene variant that indicates existence of orpredisposition to a genetic disease, either from acquired or inheritedmutations in an individual DNA. These include genetic diseases such ascystic fibrosis, diabetes, and muscular dystrophy, as well as diseasessuch as cancer (the P53 gene is relevant to some cancers), as disclosedin U.S. patent application Ser. No. 08/143,312, already incorporated byreference.

The present invention provides a substantially novel method forperforming assays on biological arrays. While specific examples havebeen provided, the above description is illustrative and notrestrictive. Many variations of the invention will become apparent tothose of skill in the art upon review of this specification. The scopeof the invention should, therefore, be determined not with reference tothe above description, but instead should be determined with referenceto the appended claims along with their full scope of equivalents.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted

1. A method for concurrently processing multiple biological chip assayscomprising the steps of: (a) providing a biological chip platecomprising a plurality of probe arrays and, surrounding the probearrays, material resistant to the flow of liquid, thereby forming aplurality of test wells, each test well defining a space for theintroduction of a sample; (b) introducing into each test well testsamples from a plurality of different patients, wherein each test samplecontains target molecules; (c) manipulating the biological chip platewith a fluid handling device that automatically performs steps to carryout reactions between target molecules in the test samples and probes ina plurality of the test wells, wherein the fluid handling devicecontrols temperature, sample handling, substrate handling and washing ofthe test wells; and (d) interrogating the probe arrays of the biologicalchip plate with a biological chip plate reader to detect reactionsbetween target molecules and probes in a plurality of the test wells togenerate assay results. 2-31. (canceled)
 32. An array assay device, saiddevice comprising: (a) a substrate receiving element for receiving asubstrate having at least on array thereon, said substrate receivingelement comprising a bottom surface; and (b) a compression element forurging said bottom surface in a direction towards a substrate whenpresent in said substrate receiving element so as to hold said bottomsurface in a fixed position relative to said substrate.
 33. The arrayassay device according to claim 32, wherein, when said bottom surface isheld in said fixed position relative to a substrate when present in saiddevice, said bottom surface ranges from about 0.1 mm to about 2 mm fromsaid substrate.
 34. The array assay device according to claim 32,wherein said bottom surface further comprises a sealing element forproducing a seal around at least one array positioned on a substratewhen held in said fixed position.
 35. The array assay device accordingto claim 34, wherein said seal is substantially vapor and fluid tight.36. The array assay device according to claim 34, wherein said sealingelement produces an assay volume of from about 10 .mu.1 to about 1000.mu.1.
 37. The array assay device according to claim 34, wherein saidsubstrate comprises a plurality of arrays and said sealing elementproduces a plurality of individual seals around each array.
 38. Thearray assay device according to claim 37, wherein each of saidindividual seals is substantially vapor and fluid tight.
 39. The arrayassay device according to claim 32, wherein said sealing element is agasket
 40. The array assay device according to claim 32, furthercomprising at least one access port.
 41. The array assay deviceaccording to claim 40, wherein said device comprises a plurality ofaccess ports.
 42. The array assay device according to claim 40, whereinsaid device comprises at least a first fluid introduction port and asecond venting port.
 43. The array assay device according to claim 40,wherein said at least one port is resealable.
 44. The array assay deviceaccording to claim 32, further comprising a removable array holder. 45.The array assay device according to claim 44, wherein said array holderis configured to be used with an array scanner.
 46. A system forperforming array assays, said system comprising: (a) an array assaydevice according to claim 32; and (b) a substrate having at least onearray.
 47. A method for performing an array assay, said methodcomprising: (a) providing an array assay device comprising: (i) a bottomsurface, (ii) a substrate receiving element for receiving a substratehaving at least on array thereon, and (iii) a compression element forurging said bottom surface in a direction towards a substrate whenpresent in said substrate receiving element so as to hold said bottomsurface in a fixed position relative to said substrate; (b) positioninga substrate comprising at least one array in said substrate receivingelement; (c) urging said bottom surface in a direction towards saidpositioned substrate using said compression element, whereby said bottomsurface is fixed relative to said substrate present in said receivingelement; and (e) contacting a sample to said at least one array.
 48. Themethod according to claim 47, further comprising producing a seal aroundsaid at least one array.
 49. The method according to claim 48, whereinsaid seal is substantially vapor and fluid tight.
 50. The methodaccording to claim 47, wherein said device comprises at least one portand said sample is introduced through said port.
 51. The methodaccording to claim 47, further comprising mixing said sample with saidat least one array.
 52. The method according to claim 51, wherein saidmixing is accomplished by an air bubble.
 53. The method according toclaim 47, further comprising retaining said substrate in an arrayholder.
 54. A method comprising, following contacting said at least onearray to a sample according to claim 47, reading said at least onearray.
 55. The method according to claim 54, where in said at least onearray is read while in the array holder of claim
 53. 56. A methodcomprising forwarding data representing a result of a reading obtainedby the method of claim 54 from a first location to a second location.57. The method according to claim 56, wherein said second location isremote from said first location.
 58. A method comprising receiving datarepresenting a result of a reading obtained by the method of claim 54.59. A method for performing an array assay, said method comprising: (a)receiving a pre-packaged substrate having at least one array in thearray assay device of claim 32 from a remote site; (b) performing anarray assay using said received array assay device; (c) removing saidpre-packaged substrate from said array assay device; and (d) readingsaid at least one array to obtain a result.
 60. The method according toclaim 59, wherein said pre-packaged substrate comprises a substrateretained in an array holder in said array assay device.
 61. A method forperforming an array assay and reading a result of said array assay, saidmethod comprising: (a) performing an array assay using the array assaydevice of claim 32 comprising a substrate having at least on arrayretained in an array holder; (b) removing said retained substrate havingat least one array from said array assay device; and (c) mounting saidretained substrate having at least one array on an array scanner so thatsaid retained substrate having at least one array may be read by saidscanner while retained in said array holder.
 62. The method according toclaim 61, further comprising reading said mounted at least one array.63. A kit for performing an assay, said kit comprising: (a) at least onearray assay device according to claim 32; and (b) instructions for usingsaid at least one array assay device in an array based assay.
 64. Thekit according to claim 63, further comprising at least one array holder.65. The kit according to claim 63, further comprising at least onearray.
 66. The kit according to claim 63, further comprising reagentsfor generating a labeled sample.
 67. The kit according to claim 63,wherein said kit further comprises a buffer.
 68. The kit according toclaim 63, wherein said kit further comprises a wash medium.